Proteins determination

The vagueness of protein determination methods causes grief for the beginning biochemist. The identical protein solution gives one result with assay A and another with assay B. Similarly, the identical assay yields different values with identical concentrations of different proteins (e.g., 1 mg/ml of BSA, ovalbumin, cytochrome). 

Prepare reagent A by dissolving 100 g NazCOa in 1 liter (final volume) 0.5N NaOH. 

Prepare reagent B by dissolving Ig CUSO4 5H2O in 100 ml (final volume) glass-distilled water. 

Turn on the spectrophotometer or colorimeter to be used and allow it to warm up as directed by the manufacturer. 

Bring the total volume of liquid in each tube to 1.0 ml by adding an appropriate amount of glass-distilled water. 

---

Typically, quantitative protein determination is done on the one hand by colorimetric or nephelometric methods, on the other hand for more difficult analytical problems by more sophisticated techniques such as high performance liquid chromatography (HPLC), gel-electrophoresis and immunoassay. However, these methods are tedious, time-consuming and expensive. 

Q Zeng, ET Smith, DM Kurtz, RA Scott. Protein determinants of metal site reduction potentials. Site directed mutagenesis studies of Clostridium pasteurianum laibredoxin. Inorg Chim Acta 242 245-251, 1996. 

As described in Chapter 2, the first complete protein structure to be determined was the globular protein myoglobin. However, the a helix that was recognized in this structure, and which has emerged as a persistent structural motif in the many hundreds of globular proteins determined subsequently, was first observed in x-ray diffraction studies of fibrous proteins. 

The Primary Structure of a Protein Determining the Amino Acid Sequence... 

Na Influx Studies. Na influx was monitored according to the procedure of Owen and Villereal (34), with some modifications. Cells were seeded onto 60-mm culture dishes, grown, and serum starved as described for the assays above. The cells were washed with incubation media and incubated in 3 ml of the appropriate agent at 37 C. After incubation the cells were rapidly washed in ice cold 0.1 mM MgCL and extracted with 5% TCA/0.5% KNO3 for sodium determination or 0.2% SDS for protein determination. Sodium concentration was measured using a Varian Model 275 Atomic Absorption Spectrophotometer. Protein was determined fluorimetrically. 

Some drugs act as Indicators (e.g. phenolphthaleln, vegetable laxatives) and affect tests carried out at a particular pH. The presence of sulfobromophthaleln dye (BSP) In serum will Interfere with serum protein determined by the biuret method. 

Protein determination Protein content was determined by Lowry s method (15) with bovine serum albumin as standard. The protein content in the coliunneluates was measured by the absorbancy at 280 nm. 

Experiments in 500 ml Erlenmeyer flasks and Fernbach flasks contained 200 ml and 1 L of EPl and EP2 medium respectively. Inocuia added to these cultures was 2 ml of spore suspension (5.0 optical density at 540 nm) for each 100 ml EP medium. All cultures were grown at 37°C in a shaking incubator (New Brunswik Sci. Co., USA), at 200 rpm. Then 10 ml of sample were withdrawn each 24 h during fermentation and immediately filtered through Millipore membranes of 0.45 pm pore size these cell-free filtrates were used for enzymatic assays and extracellular protein determinations by the Lowry method (14). Experiments in the 14 L fermentor (Microgen Fermentor New Brunswik Sci. Co., USA) were carried with lOL of fermentation medium EP2 and inoculum added was IL of mycelium grown 24 h in... 

There are many proteins in the human body. A few hundreds of these compounds can be identified in urine. The qualitative determination of one or a series of proteins is performed by one of the electrophoresis techniques. Capillary electrophoresis can be automated and thus more quantified (Oda et al. 1997). Newer techniques also enable quantitative determination of proteins by gel electrophoresis (Wiedeman and Umbreit 1999). For quantitative determinations, the former method of decomposition into the constituent amino acids was followed by an automated spectropho-tometric measurement of the ninhydrin-amino add complex. Currently, a number of methods are available, induding spectrophotometry (Doumas and Peters 1997) and, most frequently, ELISAs. Small proteins can be detected by techniques such as electrophoresis, isoelectric focusing, and chromatography (Waller et al. 1989). These methods have the advantage of low detection limits. Sometimes, these methods have a lack of specifidty (cross-over reactions) and HPLC techniques are increasingly used to assess different proteins. The state-of-the-art of protein determination was mentioned by Walker (1996). 

Karim, M.R., Shinagawa, S., Takagi, T. (1994). Electrophoretic mobilities of the complexes between sodium dodecyl sulfate and various peptides or proteins determined by free solution electrophoresis using coated capillaries. Electrophoresis 15, 1141-1146. 

Sloat AL, Roper MG, Lin X, Ferrance JP, Landers JP, Colyer CL (2008) Protein determination by microchip capillary electrophoresis using an asymmetric squarylium dye noncovalent labeling and nonequilibrium measurement of association constants. Electrophoresis 29 3446-3455... 

Domain A discrete portion of a protein with its own function. The combination of domains in a single protein determines its overall function. 

F. Ronca, Protein Determination in Polychromed Stone Sculptures, Stuccoes and Gesso Grounds, Studies in Conservation, 39, 107 120 (1994). 

Consequently, the research work of Hara s group continued focusing on the improvement of protein determination using CE combined with online CL detection. By replacing EY by the Rhodamine B isothiocyanate (RITC) dye in the binary complexes formed with the proteins BSA or human serum albumin (HSA) and using a different imidazole buffer solution of pH 6, the sensitivity was increased [72], However, best detection limits for these determinations were found employing the tetramethylrhodamine isothiocyanate isomer (TRITC) dye, left for 4 h with a standard solution of BSA in acetonitrile followed by introduction into the capillary. For BSA, a detection limit of 6 nM was reached [73],... 

Bradford reagent contains the dye Coomassie blue G-250 in an acidic solution. The dye binds to protein, yielding a blue colour that absorbs maximally at 595 nm Copper-containing reagent that, when reduced by protein, reacts with bicinchonic acid yielding a complex that displays an absorbance maximum at 562 nm Essentially involves initial precipitation of protein out of solution by addition of trichloroacetic acid. The protein precipitate is redissolved in NaOH and the Lowry method of protein determination is then performed Interaction of silver with protein - very sensitive method... 

Protein determination procedures using bicinchonic acid were developed by Pierce Chemicals, who hold a patent on the product. The procedure entails the use of a copper-based reagent containing bicinchonic acid. Upon incubation with a protein sample, the copper is reduced. In the reduced state it reacts with bicinchonic acid, yielding a purple colour that absorbs maximally at 562 nm. 

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

Balguerie, A., Dos Reis, S., Coulary-Salin, B., Chaignepain, S., Sabourin, M., Schmitter, J. M., and Saupe, S.J. (2004). The sequences appended to the amyloid core region of the HET-s prion protein determine higher-order aggregate organization in vivo. J. Cell Sci. 117, 2599-2610. 

Tanaka, M., Chien, P., Naber, N., Cooke, R., and Weissman,J. S. (2004). Conformational variations in an infectious protein determine prion strain differences. Nature 428, 323-328. 

Evolutionary processes driven by environmental changes and varying conditions have an impact on all components in a living cell. Thus, the primary, secondary and tertiary structure of proteins determines their function and location, giving different properties in different compartments, such as outer membrane, periplasmic space, cytoplasmic membrane or cytoplasm. Proteins can function as monomers or oligomers and can occur in a soluble form, as integral constituents embedded within the membrane, or can be found associated with the lipid bilayer itself or components therein. 

For N-linked chains, it is pertinent to discuss the latter aspect, for which some constraints on degrees of freedom have been established. For O-linked chains, a picture in which the chains can be said to be in intimate contact with the protein, imposing conformational constraints and allowing for joint oligosaccharide-protein determinants, is emerging. 

---